The α-amylase family, or glycoside hydrolase family 13 (Henrissat, B. and Davies, G. (1997) Curr. Opin. Struct. Biol. 7, 637-644), is a large family of starch processing enzymes. The (β/α)8-barrel fold of the catalytic domain, the catalytic site residues and the α-retaining bond cleavage mechanism are conserved in this family (McCarter, J. D. and Withers, S. G. (1994) Curr. Opin. Struct. Biol. 4, 885-892; Uitdehaag, J. C. M., et al., (1999) Nature Struct. Biol. 6, 432436), but the product and reaction specificity vary widely (Kuriki, T. and Imanaka, T. (1999) J. Biosci. Bioeng. 87, 557-565).
The α-amylase family 13 includes enzymes such as cyclodextrin glycosyltransferase—also known as cyclomaltodextrin glucanotransferase or cyclodextrin glucanotransferase (GGTase EC 2.4.1.19) and α-amylases (EC 3.2.1). CGTase and α-amylase are two classes of glycosylases that degrade starch by hydrolysis of α-(1,4)-glycosidic bonds, but the initial break down products are predominantly cyclic oligosaccharides for CGTases (also called cyclodextrins) and linear oligosaccharides for the α-amylases.
CGTases can catalyse the breakdown of starch and similar substances into cyclodextrins via an intramolecular transglycosylation reaction, thereby forming circular α(1,4)-linked oligosaccharides of varying sizes called cyclodextrins (also referred to as CDs). The circular α-(1,4)-linked oligosaccharides (cyclodextrins) are formed from linear α-(1,4)-linked oligosaccharide substrates.
The CGTase enzyme consists of five domains (A-E); domains A and B constitute the catalytic domains, domain E is involved in raw starch binding (Penninga, D., et al., (1996) J. Biol. Chem. 271, 32777-32784; Ohdan, K., et al. (2000) Appl. Environ. Microbiol. 66, 3058-3064), while the functions of domains C and D are not known yet. After binding of the substrate across several sugar binding subsites (labelled −7 to +2; FIG. 1), the α-(1,4)-glycosidic bond between subsites −1 and +1 is cleaved to yield a covalent glycosyl-enzyme intermediate that is bound at the donor subsites (−1, −2, −3, etc.) (Uitdehaag, J. C. et al., (1999) Nature Struct. Biol. 6, 432-436). In the next step of the reaction an acceptor molecule binds at acceptor subsite +1 and cleaves the glycosyl-enzyme bond.
In the cyclization reaction the non-reducing end of the covalently bound sugar is used as the acceptor to yield a cyclodextrin. At a very low rate, CGTase may also use water or a second sugar molecule as acceptor, which results in a hydrolysis or a disproportionation reaction, respectively, thus forming linear oligosaccharides.
While α-amylase is a strongly hydrolytic enzyme, CGTase is first of all a transglycosylase. The hydrolitic activity of CGTase is generally much lower than the transglycosylation activity. It has been reported that CGTases from Thermoanaerobacter and Thermoanaerobacterium thermosulfurigenes strain EM1 (Tubium) have relatively high hydrolysis activity, although still considerably lower than compared to α-amylases (Norman and Jorgensen, 1992, Denpun Kagaku, 39: 101-108; Wind et al., 1995, Appl. Environm. Microbiol. 61: 1257-1265).
It has been suggested that the relative efficiencies of the hydrolysis and transglycosylation reactions of the CGTase enzyme are determined by the nature of the acceptor used in the second half of the reaction and thus by the properties of the acceptor subsites. In respect of this, van der Veen and colleagues have reported that CGTase has a clear preference for glucosyl acceptors, as its transglycosylation activities are much higher than the hydrolysis activity (van der Veen, B. A., et al., (2000) Eur. J. Biochem. 267, 658-665). In this respect, Nakamura et al., report that +2 substrate binding subsite, which contains a conserved Phe184 and Phe260 residues is important for the transglycosylation activity (Nakamura et al., 1994, Biochemistry, 33: 9926-9936). Furthermore, Leemhuis et al., have reported that the amino acid side chain at position 260 also controls the hydrolytic activity of CGTases (Leemhius et al., 2002, FEBS letters 514: 189-192). Moreover, they have reported that mutating Phe260 can change CGTase from transglycosylase to a starch hydrolase.
U.S. Pat. No. 6,482,622, discloses that a maltogenic alpha-amylase from Bacillus, commercially available under the trade name Novamyl®, shares several characteristics with CGTases, including sequence homology and formation of transglycosylation products. CGTase variants are described that have the ability to form linear oligosaccharides when acting on starch.